Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.

Measurements of single molecular affinity interactions between carbohydrate-binding modules and crystalline cellulose fibrils.

Combining atomic force microscopy (AFM) recognition imaging and single molecule dynamic force spectroscopy (SMDFS), we studied the single molecule affinity interactions between the carbohydrate-binding module (CBM) and plant cell wall cellulose using the CBM3a (from Clostridium thermocellum) and CBM2a (from Cellvibrio japonicus) functionalized AFM tips. The binding efficiencies of the CBMs to the cellulose were determined by the binding areas on the crystalline cellulose fibrils surface using the recognition imaging. Several dynamic and kinetic parameters, such as the reconstructed free energy change, energy barrier and bond lifetime constant, were also obtained based on the measured single molecule unbinding forces, which are used to illuminate the affinity of the CBMs binding to the natural and single cellulose surface from a totally different aspect. It was found that CBM3a has a little higher binding efficiency and affinity than CBM2a to both natural and extracted cellulose surfaces and both the CBMs have higher affinities to the natural cell wall cellulose compared to the extracted single cellulose. The in-depth understanding of the binding mechanisms of the CBM-cellulose interactions of this study may pave the way for more efficient plant cell wall degradation and eventually facilitate biofuel production.

Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions.

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay "Congo Red Analysis of Cellulose Concentration," or "CRACC." CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation.

UNLABELLED: The heteropolysaccharide xyloglucan (XyG) comprises up to one-quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone-cleaving endo-xyloglucanases and side-chain cleaving exo-glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram-negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side-chain cleaving exo-glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N-terminal glycoside hydrolase family 74 (GH74) endo-xyloglucanase module in train with two carbohydrate-binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4 -based xylogluco-oligosaccharide (XyGO) substrates for downstream enzymes through an endo-dissociative mode of action. X-ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate-binding cleft specifically adapted to XyG recognition, which is composed of two seven-bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose-binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. DATABASE: Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: 5FKR, 5FKS, 5FKT and 5FKQ.

Family 6 carbohydrate-binding modules display multiple beta1,3-linked glucan-specific binding interfaces.

Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the beta-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer beta-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes beta1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to beta1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize beta1,3-glucans.